The incidence of monozygotic twinning (MZT) is higher in pregnancies conceived after assisted reproduction than after natural conception. Alterations, produced by ovarian stimulation, in-vitro culture conditions and specifically alterations of zona pellucida are mentioned as possible causes of this phenomenon. A retrospective review was performed of the incidence of MZT in pregnancies generated in our centre during the period of January 1996 to December 1999. This variable was compared in 129 gestations that resulted from blastocyst transfer (occurring from September 1998 to August 1999) with 814 pregnancies produced by transfers of 4- to 8-cell embryos. Follicular development was induced with human menopausal gonadotrophin and urinary FSH during 1996 and 1997 and with recombinant FSH during 1998 and 1999. Blastocysts were cultured in sequential media using S1 or G1 up to 72 h and S2 or G2 to day 5. Five of the 129 pregnancies generated by blastocyst transfers were complicated by MZT gestation (3.9%). In comparison, only six of 814 pregnancies occurred from 4- to 8-cell transfers (0.7%), a difference that is statistically significant (P< 0.001 with Yates correction). The results confirm an increase of MZT in pregnancies from intracytoplasmic sperm injection as compared to the natural incidence. Moreover, the frequency of MZT was significantly higher when transfers were performed at the blastocyst stage, suggesting that extended in-vitro culture of embryos may be associated with alterations of the zona pellucida and the hatching process.